Mycobacterium tuberculosis is a human pathogen that causes tuberculosis, one of the world's deadliest diseases. Current estimates are that up to one-third of the world's population may be infected with M. tuberculosis; over two million people die from TB-related diseases each year (Dye et al., 1999, JAMA 282: 677-686). Tuberculosis has resurged today, in part due to the lack of compliance to drug treatment regimes, the appearance of multiple-drug-resistant strains, and the AIDS epidemic. Consequently, increasing our understanding of the origins of pathogenicity of M. tuberculosis is a high priority of tuberculosis research.
In Mycobacterium tuberculosis, the desaturase DesA3 (encoded by gene rv3229c) is a membrane-bound stearoyl-CoA desaturase that works together with oxidoreductase Rv3230c (encoded by gene rv3230c) to produce oleic acid, an essential constituent of mycobacterial membrane phospholipids and triglycerides. Due to the physiological importance of oleic acid to bacteria, DesA3 is among the approximately 200 genes required for pathogen survival inside the granuloma enclosing a dormant tuberculosis infection. DesA3 is a target of the second-line anti-tuberculosis drug isoxyl, which has been used with isoniazid in multiple drug therapy.
To study mycobacterial pathogenicity, Mycobacterium smegmatis (M. smegmatis), a non-pathogenic strain that grows relatively faster than pathogenic mycobacteria and that transforms efficiently, has been widely used as a host for expression of target genes and proteins from pathogenic mycobacteria (Snapper et al., 1990, Mol. Microbiol. 4: 1911-1919). Many genes from pathogenic mycobacteria yield folded proteins and active enzymes when expressed in M. smegmatis, whereas the same genes expressed in Escherichia coli are neither folded nor active. The origin of this difference is not known. Currently, several different types of vectors have been developed for constitutive or inducible expression in M. smegmatis in order to access this biological capability (Blokpoel et al., 2005, Nucleic Acids Res. 33:e22; Ehrt et al., 2005, Nucleic Acids Res. 33: e21).
The inventors discovered that M. tuberculosis DesA3 expressed in M. smegmatis as a fusion with either a C-terminal His6 or a myc tag has higher catalytic activity and stability than DesA3 expressed with the natural C-terminal sequence (Chang and Fox, 2006, Biochemistry 45: 13476-13486). However, the origin of this difference was not known.
Within living cells, proteolysis is a ubiquitous and often selective process. In yeast and higher eukaryotes, some proteins are marked for degradation by modification with ubiquitin and targeted to the proteosome, and other specific routes for protein processing have been identified. Interestingly, proteolytic processing is also used to control mammalian desaturase activity (Kato et al., 2006, J. Cell Sci. 119: 2342-2353; Mziaut et al., 2000, Proc. Natl. Acad. Sci. USA 97: 8883-8888), although the proteolysis complexes used and the recognition sequences are distinct. This common approach to maintaining control of stearoyl-CoA desaturase activity serves to emphasize the importance of this key branch point in fatty acid metabolism.
Previous studies of the E. coli C-terminal targeted protein degradation systems ClpXP and Tsp showed that small and uncharged residues (Ala, Cys, Ser, Thr, Val) in last three positions of a protein enhanced the degradation (Kato et al., 2006, J. Cell Sci. 119: 2342-2353; Keiler and Sauer, 1996, J. Biol. Chem. 271: 2589-2593). In E. coli and other bacteria, several types of C-terminal targeted protein degradation complexes have been identified, including the ClpXP, ClpAP, FtsH (HfIB), and Tsp (Prc) systems as major contributors to protein degradation in E. coli (Gottesman et al., 1998, Genes Dev. 1338-1347; Herman et al., 1998, Genes Dev. 12: 1348-1355; Keiler and Sauer, 1996, J. Biol. Chem. 271: 2589-2593). Among these, the Clp proteases are cytoplasmic, FtsH is a membrane protease, and Tsp is a periplasmic protease, ensuring that tagged proteins can be degraded in all cellular compartments. Thus the known proteolytic systems have different substrate recognition properties, and different contributions to proteolytic processing.